Summary of work: There are two unique members of the hsp70 heat-shock protein family in male germ cells. The goals of these studies are to identify the mechanisms regulating their expression and to determine the roles of these proteins in germ cells. The HSP70 proteins are molecular chaperones that assist in the folding of nascent polypeptides and assembly of multimeric complexes, and in the refolding of denatured proteins following heat shock and other stresses. The genes for most HSP70 proteins are expressed constitutively (Hsc70, Hsp78) or in response to stress (Hsp70-1 and Hsp70-3), while Hsp70-2 and Hsc70t are expressed in response to developmental cues and only in male germ cells. Permanent cell lines are not available for promoter analysis studies of gene expression in male germ cells and reliable methods for transfecting primary cultures of germ cells have not been developed. We therefore used transgenic mice to delimit the upstream regulatory region required for developmental expression of Hsp70-2. Different Hsp70-2 gene promoter fragments were ligated to the Lacz reporter gene and beta-galactosidase expression determined in the testes of transgenic mice. It was found that sequences within 604 bp of the translation start site are required for correct expression. This region was examined further with in vitro methods. Footprint analysis identified two domains protected from DNase digestion by germ cell nuclear proteins, referred to as box 1 (between bp -555 and -503) and box 2 (between bp -346 and -335). These domains contain clusters of transcription factor binding motifs. Gel shift and super-shift analyses indicated that several known transcription factors and unknown proteins present in germ cell nuclei bind to specific sequences in these regions. HSP70-2 is synthesized during the meiotic phase of male germ cell development and we hypothesized that it is a chaperone for proteins involved in meiosis. This was confirmed using the gene knockout approach. Disruption of the Hsp70-2 gene resulted in developmental arrest and apoptosis of pachytene spermatocytes at the G2/M-phase transition of meiosis I. Since this event requires cyclin B1-dependent Cdc2 kinase activity, we further hypothesized that HSP70-2 is a chaperone required for Cdc2 activation. Although Cdc2 was present in the testis of Hsp70-2 knockout mice, it did not form a heterodimer with cyclin B1 and lacked kinase activity. Addition of recombinant HSP70-2 protein to a homogenate of testis from Hsp70-2 knockout mice restored the ability of Cdc2 to form a heterodimer with cyclin B1 and to become an active kinase, confirming that HSP70-2 is a chaperone for Cdc2. However, the effect is leaky and a few germ cells avoid apoptosis, undergo one or sometimes both meiotic divisions and begin acrosome formation. This indicates that some developmental events that normally occur in spermatids do not require completion of meiosis. Similar results occur in Drosophila with mutations in the Cdc25 or Cdc2 genes, suggesting that other kinases may partially compensate in the absence of Cdc2 kinase activity. The HSC70T protein is present only in spermatids, during the post-meiotic phase of male germ cell development. By analogy with HSP70-2, we hypothesized that HSC70T is a chaperone for unique proteins involved in post-meiotic germ cell development or sperm function. However, male Hsc70t knockout mice have normal fertility and there are no apparent changes in testis morphology or in sperm numbers and motility. The absence of an overt phenotype suggested that other HSP70 proteins might compensate for the absence of HSC70T, but no changes in their mRNA or protein levels were found by northern or western blot analysis. However, closer examination found that sperm from Hsc70t-/- mice incubated for longer than 30 minutes in vitro because immotile, while those from wild-type retained motility for several hours. ATP levels of sperm from Hsc70t-/- mice is about 1% of that in wild-type sperm and cAMP levels are about half. Preliminary results indicate that conversion of lactate to pyruvate is compromised, suggesting that LDH-C function is altered or that NADH levels are reduced in sperm from Hsc70t-/- mice. Yeast two-hybrid screens identified a BAG-domain protein that binds to HSC70T and HSP70-2 and is expressed predominantly in testis. The protein binds through its BAG domain to the N-terminal ATPase domain of HSC70T and HSP70-2 and inhibits their ATPase activity. The human homolog was reported to bind to the death domain on some tumor necrosis factor-" receptors (TNFR). It was named "silencer of death domain" (SODD) and found to bind to HSC70. SODD was hypothesized to regulate apoptosis by suppressing signal transduction events downstream of death-domain containing proteins such as TNFR1. However, we made the significant finding that TNFR1 has an atypical ATPase domain to which SODD binds. Recent studies indicate that SODD modulates TNFR1 function by inhibiting the ATPase activity required for receptor clustering and activation of the apoptosis cascade. Significance: Spermatogenic cells contain two unique HSP70 proteins, but are particularly susceptible to damage by elevated temperature and environmental agents and must be maintained a few degrees below body temperature to develop. This suggests that HSP70-2 and HSC70T have evolved for purposes other than to protect against heat shock or stress and have unique chaperone roles in spermatogenesis. Identifying these roles, determining how their function is regulated, and learning the mechanisms which are sensitive to damage may lead to novel strategies for prevention, diagnosis or treatment of the effects of reproductive toxicants or other causes of infertility, or to the future development of a male contraceptive.